Preferential stimulation of melanocytes by M2 macrophages to produce melanin through vascular endothelial growth factor

Post-inflammatory hyperpigmentation is a skin discoloration process that occurs following an inflammatory response or wound. As the skin begins to heal, macrophages first exhibit a proinflammatory phenotype (M1) during the early stages of tissue repair and then transition to a pro-healing, anti-inflammatory phenotype (M2) in later stages. During this process, M1 macrophages remove invading bacteria and M2 macrophages remodel surrounding tissue; however, the relationship between macrophages and pigmentation is unclear. In this study, we examined the effect of macrophages on melanin pigmentation using human induced pluripotent stem cells. Functional melanocytes were differentiated from human induced pluripotent stem cells and named as hiMels. The generated hiMels were then individually cocultured with M1 and M2 macrophages. Melanin synthesis decreased in hiMels cocultured with M1 macrophages but significantly increased in hiMels cocultured with M2 macrophages. Moreover, the expression of vascular endothelial growth factor was increased in M2 cocultured media. Our findings suggest that M2 macrophages, and not M1 macrophages, induce hyperpigmentation in scarred areas of the skin during tissue repair.


Results
Differentiation of melanocytes from hiPSCs. To generate hiMels, we adapted protocols from other reports 7,28 . Peripheral blood mononuclear cell-derived hiPSC lines were used in this study. A schematic of the melanocyte differentiation protocol is shown in Fig. 1a. We observed morphological changes during differentiation (Fig. 1b). hiPSCs showed embryonic stem cell-like colonies during expansion. On day 28, the cell morphology changed to spindle-shaped cells with black pigmentation. Compared to hiPSC pellets, hiMel pellets exhibited a relatively dark shade on day 28 and were visible in the cells (Fig. 1c). Moreover, mature melanosomes were observed in the cytoplasm of hiMels (Fig. 1d). To confirm whether hiMels exhibited the characteristics of human epidermal melanocytes (NHEMs), we confirmed the expression of specific markers. NHEMs (darkly pigmented donor) were used as a positive control (Fig. 1e). The level of the pluripotency marker OCT4 was significantly reduced in differentiated cells and NHEMs. Positive expression of the melanocyte-specific markers PAX3, SOX10, MITF, TYRP1, and TYR was increased in hiMels. The differentiated hiMels were also positive for Fontana-Masson and L-DOPA staining but not for hiPSCs ( Fig. 1f-h). Immunocytochemistry confirmed that expression of the mature melanocyte marker TRP1 was increased in hiMels compared to in hiPSCs (Fig. 1i). Tyrosinase activity measured at OD 475 nm indicated biosynthesis of melanin by hiMels (Fig. 1j). Together, these data demonstrate that hiPSCs can differentiate into functional melanocytes.
Coculture of hiMels with M1 or M2 macrophages. To provide inflammatory and tissue repair environments, we cocultured hiMels with M1 or M2 macrophages (Fig. 3a). The cytokines secreted by the cells were predicted to be exchanged through the transwells. After 1, 3, and 5 days of coculture, melanin content was measured in cocultured hiMels (Fig. 3b). After 5 days, hiMels cocultured with M2 exhibited significantly increased melanin biosynthesis compared to hiMels cocultured with MΦ. However, hiMels cocultured with M1 showed no significant increase in the melanin content compared to the two groups. Figure 3c provides a whole image of the hiMel-coculture plate before staining. L-DOPA staining of cocultured hiMels showed a similar tendency as the measured melanin content (Fig. 3d). hiMels cocultured with M2 synthesized more melanin in the cytoplasm compared to hiMels cocultured with MΦ; however, hiMels cocultured with M1 synthesized less melanin compared to hiMels cocultured with MΦ (Fig. 3e). Expression of the melanocyte pigmentation markers MITF, TYRP1, TYR , DCT, and MLANA was quantified in cocultured hiMels (Fig. 3f). Marker expression was significantly lower in hiMels cocultured with M1 than in hiMels cocultured with MΦ. hiMels cocultured with M2 showed increased levels of pigmentation markers compared with hiMels cocultured with M1. These results confirm that the proinflammatory environment induced by M1 macrophages decreased melanin biosynthesis in hiMels. In contrast, melanin biosynthesis was increased in hiMels cocultured with M2, indicating that the tissue repair environment induces melanin biosynthesis. These findings suggest that skin pigmentation is affected by the in vivo environment, specifically by resident macrophages.
Cytokine evaluation in coculture media of hiMels and macrophages. To identify cytokines secreted from macrophages that affect melanocyte pigmentation, a cytokine array was performed in the con- www.nature.com/scientificreports/ ditioned media of each hiMel and macrophage group (Fig. 4a). The levels of the inflammatory factors IP-10, CXCL9, IL-6, and CXCL5 in the hiMel-M1 group were higher than those in the hiMel-M2 group (Fig. 4b). The levels of the pro-angiogenesis factor TIM-3 was higher in the hiMel-M2 group than in the other groups. Moreover, the hiMel-M2 group showed increased secretion of the angiogenic factors VEGF and CD31 compared to in the hiMel-M1 group (Fig. 4c). The level of IP-10, which is secreted by M1 macrophages, was approximately 20-fold higher than that in other groups, and that of VEGF, which is secreted by M2 macrophages, was 25-fold higher than that in the hiMel-M1 group (Fig. 4d), suggesting that IP-10 secreted by M1 macrophages and VEGF secreted by M2 macrophages affected the pigmentation of hiMels in the coculture platform.
Inhibition of VEGF reduced melanin production in cocultured hiMels with M2 macrophages. As shown in Fig. 4, VEGF was selected as a candidate affecting melanin biosynthesis in cocultured hiMels. To confirm this result, hiMels cocultured with M2 were treated with 120 ng/mL human VEGF165 antibody for 5 days of coculture (Fig. 5a). After coculture, the melanin content was measured in cocultured hiMels (Fig. 5b). hiMels cocultured with M2 showed upregulated melanin production compared to those cocultured with MΦ; however, this production was downregulated by neutralization of VEGF. VEGF neutralization also reduced melanogenesis in hiMels cocultured with M2 as confirmed by L-DOPA staining (Fig. 5c,d). Moreover, the tyrosinase activity assay of M2 cocultured hiMels with or without antibody showed similar levels of L-DOPA staining (Fig. 5e). www.nature.com/scientificreports/ Quantitative reverse transcription-PCR analysis of the MITF, TYRP1, TYR , DCT, and MLANA gene expression levels was performed in 5-day cocultured hiMels with or without VEGF antibody (Fig. 5f). In hiMels cocultured with M2, the expression of TYRP1 and TYR mRNA was significantly increased compared to that in hiMels cocultured with MΦ, and hiMels cocultured with M2 and VEGF antibody showed significantly decreased MITF, TYRP1, TYR , DCT, and MLANA mRNA levels compared to without VEGF antibody. According to the western blotting results, the protein levels of the MITF, TYR, and TRP1 markers were similar to those in other experiments ( Fig. 5g,h). These results suggest that VEGF secreted from M2 macrophages promoted melanin synthesis in the in vitro coculture system.

Changes in melanin biosynthesis by VEGF in hiMels.
To confirm the previous results, hiMels were treated VEGF. Before treatment, the CCK assay was performed to determine whether VEGF affected hiMel proliferation ( Supplementary Fig. S2). VEGF (0-50 ng/mL) was not cytotoxic to hiMels after 72 h of treatment. Treatment with VEGF (20 ng/mL) activated melanin biosynthesis and melanogenesis according to L-DOPA staining (Fig. 6a). VEGF-treated hiMels showed higher percentage of pigmented areas than that seen with the control (Fig. 6b). The melanin content assay and tyrosinase activity assay also showed that melanin levels increased in VEGF-treated hiMels compared to in controls (Fig. 6c,d). Expression of mature melanocyte mark- www.nature.com/scientificreports/ ers, such as MITF, TYRP1, TYR , and MLAMA, was upregulated by VEGF treatment, and most markers appeared to increase in a dose-dependent manner (Fig. 6e). Moreover, we confirmed that MITF, TYR, and TRP1 were more abundant in VEGF-treated iMels than in controls using western blot analysis ( Fig. 6f,g). These results suggest that hiMels reacted with the selected cytokines and that the previous results were related to VEGF secreted by M2 macrophage types.

Discussion
Skin pigmentation is a complication that typically occurs during scar inflammation after surgical procedures or burns 30 . When the skin is damaged, immune cells migrate to remodel the wound site; however, newly formed skin tissue may exhibit a different color compared to the original tissue 31 . Hyperpigmentation occurs because of high levels of inflammation in the scar 32 . This visible change in the skin during inflammation raises cosmetic and psychosocial concerns for the patient.
Macrophages play a critical role in innate immunity. When inflammation breaks because of scar formation, macrophages first migrate to the wound 14,17 . Upon reaching the wound site, M1 macrophages begin to secrete inflammatory cytokines such as IL-1β, IL-6, and IL-12 and remove bacteria or viruses that have entered the body 15,16 . During this process, the wounded area tends to become swollen, damaging surrounding cells. Once the foreign substances are removed, M2 macrophages migrate to the site and secrete anti-inflammatory cytokines such as IL-10, TGF-β1, and VEGF to remodel the surrounding tissues 33,34 . Skin pigmentation occurs during this process, which can sometimes persist for a long period 35 . The relationship between the wound healing process and scar pigmentation is not well-understood; therefore, continuous research on this topic is needed. Direct studies of melanocytes are complicated because of the lack of a source of these cells; thus, further research using a new cell source is required.
To overcome this issue, research using hiPSCs as a cell source has been actively conducted 36,37 . Importantly, iPSC technology can be used in studies requiring cells that are difficult to obtain, including melanocytes. Mica et al. differentiated hiPSCs into melanocytes through neural crest generation 7,28 . hiPSCs plated on www.nature.com/scientificreports/ Matrigel differentiated into neural crests, which were transferred onto fibronectin-coated plates as droplets, and Chir99021, BMP4, and EDN3 were used to generate mature melanocytes. In this study, pigmentation of hiMels was visually confirmed in the cell pellets (Fig. 1c). Gene expression of melanocyte markers increased in hiMels, whereas pluripotency marker expression was decreased (Fig. 1e). We further confirmed that hiMels were positively stained using Fontana-Masson and L-DOPA staining, which are melanin-specific ( Fig. 1f-h).
Travis et al. observed the process of hyperpigmentation during wound healing in swine models 31 . They reported that α-MSH, which has potent anti-inflammatory effects, increases pigmentation by regulating the expression of proinflammatory and anti-inflammatory cytokines 38 . This study suggests a close relationship between skin pigmentation and inflammation. Based on other studies, we attempted to confirm the effect of macrophages on pigmentation using a coculture platform. We differentiated M1 and M2 macrophages from THP-1 monocytes to mimic the human immune system in vitro. THP-1 monocytes were differentiated into MΦ and then polarized into M1 and M2 macrophages. We confirmed the gene expression levels of macrophage markers. The levels of inflammatory markers, such as CXCL10, IL-6, IL-1β, and IL-12, were increased in M1 macrophages (Fig. 2b). In contrast, the expression of wound healing markers, such as CD163, CD206, IL-10, and VEGF, was higher in M2 macrophages (Fig. 2c). Expression of the M1 cytokine IL-6 and M2 cytokine TGF-β was also confirmed (Fig. 2d). We used an in vitro coculture system to investigate the interaction between macrophages and pigmentation. After 5 days of coculture, melanin levels were low in the inflammatory environment and high in the wound healing environment (Fig. 3b). A similar tendency was observed with L-DOPA staining (Fig. 3d). The levels of the pigmented area increased in M2 cocultured hiMels, whereas M1 cocultured hiMels showed lower levels compared to M0 cocultured hiMels (Fig. 3e). We also confirmed the gene expression levels of pigmentation markers (Fig. 3f).The gene expression levels of pigmentation markers were significantly decreased in the inflammatory environment and increased in the wound healing environment induced by different macrophage types. Based on these results, inflammation reduces melanocyte pigmentation, whereas the tissue repair environment promotes melanocyte pigmentation.
To identify the specific cytokines affecting pigmentation in each environment, a human cytokine array was performed with the coculture media. Inflammatory cytokines were generally detected in the hiMels-M1 group and IP-10 levels were significantly increased (Fig. 4b). High levels of cytokines related to tissue repair were detected in the hiMel-M2 group (Fig. 4d). The VEGF level was higher in the culture supernatant of the hiMel-M2 www.nature.com/scientificreports/ group than in the hiMel-M1 group. We listed M1/M2 and M2/M1 ratios in descending order (Fig. 4e). VEGF, which showed the greatest difference, was selected for neutralization in the coculture system to confirm the pigmentation changes induced by macrophages. As a result, melanogenesis was significantly reduced by the VEGF antibody in hiMels cocultured with M2 (Fig. 5). In addition, we directly treated hiMels with VEGF and verified the changes in melanin production. VEGF treatment increased pigmentation in the melanin content assay and tyrosinase activity assay (Fig. 6c,d). Gene expression and protein expression of pigmentation markers was altered, similar to the results of coculturing cells in VEGF-treated hiMels ( Fig. 6e-g). Previous studies have shown that tyrosinase protein size can be either glycosylated or deglycosylated 39,40 ; thus, in the western blot results, we observed two different forms of tyrosinase bands (Figs. 5g, 6f, respectively). The deglycosylated form of tyrosinase was detected at 60 kDa (Fig. 5g), whereas the glycosylated form appeared at 80-90 kDa (Fig. 5g). These results www.nature.com/scientificreports/ are thought to be because of the distinct differentiation efficiency of iPSCs and iMels. It is hypothesized that even if the differentiation process or the harvest timepoints are the same, the degree of iMel maturity can be different. Moreover, it can be also speculated that tyrosinase was deglycosylated during coculture with macrophages. The effects of VEGF on melanocytes have been reported in several studies. Zhu et al. showed that VEGF enhanced tyrosinase activity and melanin production 27 . Regazzetti et al. attempted to confirm the effect of endothelial cells on melanocytes because they observed that vascularization affected skin pigmentation in patients with benign vascular skin lesions 41 . By coculturing human dermal microvascular endothelial cells with human melanocytes, the authors suggested that VEGF is involved in melanogenesis and melanocyte proliferation. Although the effect of VEGF secreted by M2 macrophages on melanocytes or pigmentation has not been evaluated, our study suggests that this secretion affects melanocyte activity or hyperpigmentation during inflammation in the skin tissue.
There were several limitations to our study. Our in vitro system does not fully mimic the in vivo environment, as the inflammation process typically requires both M1 and M2 in the same environment. Further studies of the effect of VEGF as a key cytokine and confirmation of the related pathways causing melanocyte pigmentation through these factors are needed. Macrophages produce many other factors that regulate melanin synthesis. Slominiski et al. confirmed that pro-opiomelanocortin produced by macrophages upregulates melanogenesis 42,43 . Additionally, inflammatory factors such as TNF, IL-1β, and IFN-γ 23 were reported to inhibit melanin synthesis, whereas cytokines such as IL-18 25 and GM-CSF upregulate melanogenesis 20 . Confirmation of these proteins using our platform would be interesting as further studies. We measured melanin content as the optical density at 405 nm 44,45 ; however, several studies have suggested that this is not a direct measurement of the actual amount of melanin. More accurate measurements of the melanin content using high-performance liquid chromatography www.nature.com/scientificreports/ or electron paramagnetic resonance methods are needed [46][47][48] . We hope to confirm our results using these methods in future studies.
In conclusion, we showed that M1 and M2 macrophages decreased and increased melanin synthesis in melanocytes, respectively. VEGF expression showed the greatest increase in M2 coculture culture media. Thus, melanin production may be suppressed by cytokines secreted by M1 macrophages during the early stages of inflammation. The increased expression of VEGF by M2 macrophages may increase pigmentation and induce hyperpigmentation in the scarred area during the tissue repair process. Therefore, VEGF is a potential target for regulating skin pigmentation. Methods hiPSC culture. All hiPSC lines used in this study were generated using peripheral blood mononuclear cells.
Reprogramming and characterization were performed as previously described 49,50 . The cells were maintained in vitronectin-coated dishes (Thermo Fisher Scientific, Waltham, MA, USA), and the media were changed daily with fresh Essential 8 (E8) medium (Thermo Fisher Scientific).
Briefly, 5 × 10 4 human hiPSCs were plated on a Matrigel-coated 12-well in E8 medium containing Rho-associated protein kinase (ROCK inhibitor, 10 mM; Y-27632, PeproTech, Rocky Hill, NJ, USA). The cells were allowed to reach 70% confluence over 2-3 days with daily media changes. Differentiation was initiated by switching the medium to knockout serum replacement medium supplemented with LDN193189 (500 nM; PeproTech) and SB431542 (10 mM; Tocris, Bristol, UK). Treatment with CHIR99021 (3 mM; PeproTech) was started on day 2 of differentiation, and LDN193189 and SB431542 were withdrawn on days 3 and 4, respectively. On day 4, knockout serum replacement medium was gradually replaced with increasing amounts of N2 medium. The cells were additionally treated with human recombinant bone morphogenetic protein 4 (BMP4, 25 ng/mL; R&D Systems, Minneapolis, MN, USA) and endothelin 3 (EDN3, 100 nM; Tocris) beginning on day 5 of differentiation. The cells were collected on day 8 for passaging, dissociated, and plated on fibronectin-coated plates in full Mel medium containing CHIR99021 (3 mM), BMP4 (25 ng/mL), and EDN3 (100 nM). The media were changed on alternate days and the cells were passaged weekly for maintenance and expansion. For cytokine treatment, 1 × 10 5 melanocytes were plated into 12-well dishes. After overnight incubation, the medium was changed to differentiation medium containing the indicated concentrations of VEGF (PeproTech).
Transmission electron microscopy. Cell pellets were fixed in 2.5% glutaraldehyde in phosphate-buffered saline (PBS; pH 7.2) at 4 °C and then conventionally embedded and sectioned. The slices were viewed using a transmission electron microscope (JEOL-JEM 1010, JEOL Co., Ltd., Tokyo, Japan).
Polymerase chain reaction. Total mRNA was extracted using TRIzol reagent (Thermo Fisher Scientific).
cDNA was synthesized using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Quantitative real-time PCR was performed using SYBR Green Mix and a LightCycler 480 Instrument II (Roche). The results were calculated using the LightCycler (Roche Diagnostics). Relative gene expression was calculated using the delta cycle-threshold (ΔCt) method, and the ΔCt values were converted to ratios 51,52 . The primer sequences are presented in Supplementary Table S1 Western blot analysis. The protein was extracted from the cells as described for the tyrosinase activity assay. Proteins were separated using 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked with PBS containing 0.1% Tween 20 (PBST) and 3% BSA for 1 h and then incubated with an anti-MITF antibody (1:1000, ab12039; Abcam), antityrosinase antibody (1:500, sc-20035; Santa Cruz Biotechnology, Dallas, TX, USA), and anti-TRP1 antibody (1:1000, ab235447; Abcam) at 4 °C. The next day, membranes were washed 3 times with 0.1% PBST and incubated for 1 h with the respective secondary antibodies. After 4 washes with 0.1% PBST, the protein-of-interest was detected using a Bio-Image Analysis System (Amersham Imager 600; Fuji Photo Film Co., Ltd., Tokyo, Japan).

Inflammatory cytokine array.
A human XL cytokine array kit (ARY022B, R&D Systems) was used according to the manufacturer's protocol. Briefly, the membranes were blocked for 1 h and then incubated with coculture media overnight at 4 °C. The detection antibody cocktail was diluted in 1 × array buffer and incubated for 1 h at 25 °C. Images were obtained using the Bio-Image Analysis System (Amersham Imager 600). The intensity of the spots was quantified using ImageJ software (National Institute of Health, Bethesda, MD, USA).

Cell viability assay.
To confirm the viability of the melanocytes, we performed a cell counting Kit-8 assay (CCK-8; CK04-13, Dojindo Molecular Technologies, Kumamoto, Japan). The culture media supernatant was transferred to a 96-well plate after 72 h, and 10 μL CCK-8 solution was added to each well. Absorbance was measured at 450 nm using a microplate reader.

Statistical analysis.
All experiments were repeated at least three times. Statistical analyses were performed using GraphPad Prism 5 software (GraphPad, Inc., La Jolla, CA, USA). The results are presented as the mean and standard error of the mean. Error bars represent the standard error of the mean. Differences between groups were examined for statistical significance using Student's t test. A t test was used to analyze nonparametric quantitative datasets, and a one-tailed p-value was calculated. One-way analysis of variance, followed by Dunnett's multiple comparison test, was used for several analyses (*p < 0.05, **p < 0.01, and ***p < 0.001 indicate statistical significance).